Acute and chronic wounds are a significant burden to the healthcare system affecting millions of patients nationally and resulting in billions of dollars of outlays. Every year several million people suffer irreversible damage or loss of one or more tissue or organ systems, resulting in disfigurement, loss of function, physiologic derangement, and death.
A major leap forward in reconstructive surgery occurred in the 1960's and continued through the following decade with the discovery of “axial flaps”. Using detailed anatomic analysis, reconstructive surgeons determined that large portions of composite tissues (e.g., skin and muscle, skin and bone, skin and fascia etc.), designated “flaps” could be reliably moved from one portion of the body (donor) to an adjacent area (recipient). The development of microsurgery was the next major breakthrough. Now with the aid of an operating microscope, surgeons could reliably suture arteries and veins as small as 1 mm in diameter with patency rates over 95%. Whereas previously donor tissues could only be moved as far as their vascular “leash” would allow, microsurgical techniques have enabled reconstructive surgeons to move tissues from and to anywhere in the body, as long as recipient vessels (to which the flap artery and vein could be sewn) are found.
Despite the numerous benefits associated with tissue transfer, there are certainly drawbacks associated with these complex reconstructive procedures. Harvesting the donor tissue is a complicated process and the resultant donor sites are often painful and carry substantial morbidity, especially in the diabetic population. Patients who are elderly or carry other co-morbidities such as cardiopulmonary or renal disease are especially at risk for significant peri-operative complications because of the long anesthesia times, notable blood loss, and large fluid shifts.
If suitable replacement tissue could be fabricated with its own microvascular network and vascular leash (for attachment to host vasculature), it will be possible to create “off the shelf constructs” which could then be anastomosed to the required area of deficiency in a surgically reliable, safe, and expeditious manner.
Although the field of tissue engineering holds great promise for the fabrication of functional implantable materials (Langer et al., Science 260:920-926 (1993)), the ability to design artificial tissue constructs that have their own inherent vascular network remains a critical limiting step (Saltzman et al., Nat Rev Drug Disc 1:177-186 (2002)). Without such a network, any implanted engineered tissue must rely upon host vessel ingrowth for vascularization. However, because of the slow pace of vascular ingrowth, tissue engineers are constrained to designing constructs no thicker than a few millimeters, the limit of diffusion of nutrients from a vascular to a non-vascular area (Androjna et al., Tissue Engineering Part A 14:559-569 (2008)). Diffusion of nutrients is highly limited and, therefore, restricts the size and thickness of artificial tissue implants. In vivo, most cells cannot survive more than a few hundred micrometers away from the nearest capillary (Frerich et al., Int J Oral Maxill of 30:414-420 (2001); Okano et al., Cell transplant 7:435-442 (1998); Sheridan et al., J Control Release 64:91-102 (2000)). Cells distant from a vascular area are prone to ischemic death because of a paucity of oxygen and nutrients required for cellular metabolism.
Moreover, to be clinically useful, a tissue engineered construct must have not only a 3D vascular network, but also the ability to be “spliced” into the host blood supply, allowing for immediate vascularization of the construct and assuring the survival of the cellular constituents within. The creation of such vascularized constructs is currently beyond the capabilities of any contemporary tissue engineering approach. To rectify this problem, several investigators have endeavored to create a de novo vascular network, which could then be seeded with cells (Takei et al., Biotechnology Progress 23:182-186 (2007); Kannan et al., Biomaterials 26:1857-1875 (2005); Lim et al., Lab on a Chip 3:318-323 (2003); and McGuigan et al., Proc Nat'l Acad Sci 103:11461-11466 (2006)). Because the construct would be “pre-vascularized”, it would theoretically be able to pass oxygen and nutrients into the deeper portions of the structure. However, in this scenario, the vascular network would have to be immediately perfused by the host's blood in order for the deeper portions of the construct to survive. This would only be possible if the micro vascular network coalesced into larger vessels that could be reliably (>1 mm in diameter) anastomosed to the host's blood supply. Unfortunately, despite tremendous advances in the understanding of vasculogenesis and angiogenesis, including the creation of capillary-like networks from cellular constituents, investigators have thus far been unable to achieve the goal of creating a confluent de novo microvascular and macrovascular network. Furthermore, the vascular networks that have been constructed remain microscopic in scale, and have not yet been integrated into appropriate tissue engineering scaffolds. In short, the organizational process underlying the development of tissues and organs thus far remains too complex to engineer from “the ground up.”
Numerous investigators have attempted to engineer various constructs by placing different cell types on a variety of (3-D) matrices. Current therapeutic strategies rely upon implantation of these tissue-engineered constructs into the host where they will be re-vascularized by host vessel in-growth. Unfortunately, this is a highly fallible approach since constructs containing cells further than several hundred microns from the surface (the limits of diffusion) are prone to ischemic cell death because of a paucity of oxygen and nutrients required for cellular metabolism. This technological hurdle has significantly stunted the development of tissue engineered products that may be widely applied in the clinical arena. As an example, two of the most “successful” tissue engineered products, Integra™ and Alloderm™, developed primarily as skin substitutes, are both thin (<2 mm thick), acellular and require vascularization from the host in order to “survive”. Because of their initial avascularity, both of these constructs are prone to infection and can only be placed in healthy, well vascularized wound beds.
An alternative and much more promising strategy for tissue engineering vascularized constructs would not rely on the assemblage of a (large) construct starting from masses of individual cells. Instead, the ideal strategy would be to build a construct that already contained a vascular “scaffold”, with a microvascular bed that coalesced into a “feeding” macrovascular (>1 mm) leash. Such a construct would be ideal as it could be anastomosed to the host blood supply using standard microsurgical techniques, resulting in an immediately perfused, and thus fully viable construct. A variant of this approach has been pursued in a porcine model where a vascularized bladder-like tissue has been successfully created (Schultheiss et al., J. Uro. 173:276-280 (2005)). Their method involved de-cellularizing a segment of porcine intestine, then re-perfusing it with endothelial precursors which then re-endothelialized the intact vascular “scaffold”. The constructs were then anastomosed to recipient pigs and demonstrated normal blood flow within the re-endothelialized vessels up to one hour post-implantation.
Although these data demonstrate a successful “proof of concept”, the utility of this approach is limited by the need for a donor animal to provide the tissue, which must then be chemically treated to remove all cellular constituents. Furthermore, their constructs could not be custom designed by tissue type or shape, attributes that will likely be required when constructing any “replacement part”, other than the flat sheet they utilized as a bladder wall replacement.
In general, the methods used for tissue scaffolds range in complexity from forming porous polymeric structures (Chen et al., Macromol. Biosci. 2:67-77 (2002) and Mikos et al., Electronic Journal of Biotechnology vol. 3 (2000)) (with random hole sizes and positions) to completely designing the construct structure using 3D printing or microfabrication-based technologies (Mironov et al., Trends in Biotech 21:157-161 (2003); Khademhosseini et al., Biomaterials 28:5087-5092 (2007); and Cooke et al., Journal of Biomedical Materials Research Part B—Applied Biomaterials 64B:65-69 (2003)). Several sacrificial techniques have been used to pattern simple microfluidic networks in scaffolds (Golden et al., Lab on a Chip 7:720-725 (2007) and Nazhat et al., Biomacromolecules 8:543-551 (2007)). To date, none of these techniques have met the necessary dual criteria of providing physiologic flow through 3D microchannel networks with a capability of being surgically integrated into the host macrovasculature.
Thus, comprehensive vascularization of tissue in vitro is a major challenge in fabrication of bioengineered tissue. Creating a proper connection between the engineered tissue and the host vasculature when the tissue is implanted is the next major challenge.
Constructs with three dimensional microfluidic networks have many potential applications, including fluid mixing (Therriault et al., Nat Mater 2:265-271 (2003)), providing healing agents in self-healing polymer systems (Toohey et al., Nat Mater 6:581-585 (2007)), and artificial vascular networks for engineered tissue (Borenstein et al., Biomedical Microdevices 4:167-175 (2002); Shin et al., Biomedical Microdevices 6:269-278 (2004); Choi et al., Nat Mater 6:908-915 (2007); and Saltzman et al., Nat Rev Drug Disc 1:177-186 (2002)). However, three dimensional (3D) fluidic network fabrication is currently time-consuming and difficult, requiring either layer-by-layer assembly of two dimensional (2D) structures formed using standard microfabrication techniques (such as photolithography and imprint lithography) (Luo et al., Lab on a Chip 8:1688-1694 (2008); Chiu et al., Proc. Natl. Acad. Sci. U.S.A. 98:2961-2966 (2001); McDonald et al., Acc. Chem. Res. 35:491-499 (2002)) or 3D printers (Therriault et al., Nat Mater 2:265-271 (2003); Toohey et al., Nat Mater 6:581-585 (2007); and McDonald et al., Acc. Chem. Res. 35:491-499 (2002)).
The present invention is directed to overcoming these and other deficiencies in the art.